Project Summary/Abstract Development of antibiotic resistance by bacterial pathogens poses an inherit need for the identification of new chemical entities that will serve as new classes of antibiotics. Lanthipeptides are a family of ribosomally synthesized post-translationally modified peptides (RiPPs) consisting of structurally diverse natural products many of which are antibiotics. In this proposal, a genome mining strategy for the identification of new natural product RiPPs will be explored. The recent discovery of a new class of truncated LanB enzymes, herein termed short LanBs (sLanB), has prompted investigation of the natural products they produce. Given the rich history of lanthipeptides, formerly known as lantibiotics, as antimicrobial compounds, the natural products derived from sLanB enzymes are expected to possess similar therapeutic value and structural diversity. The first aim of this proposal involves the isolation of a natural product from a model gene cluster in Pseudomonas syringae containing a single sLanB gene. The enzymes encoded by the gene cluster will be studied by heterologous expression in E. coli. After determining the natural product structure, isolation from the native organism will be attempted by chemical derivatization. Chromosomal disruption by homologous recombinations represents an alternative method for natural product determination by isolation of advanced biosynthetic intermediates. Building upon our initial studies, the second aim of this proposal involves identification of a natural product from a complex gene cluster in Desmospora sp. 8437, containing a total of seven sLanB enzymes. The enzymes encoded by the gene cluster will be heterologous expressed in E. coli. In vivo co-expression of all seven sLanBs with tagged precursor peptide will allow for rapid determination of enzyme activity. The modifications by the sLanB enzymes will be analyzed by mass spectrometry. This overall strategy is expected to allow for rapid isolation of natural products derived from multiple sLanB enzymes. Natural products isolated from these two gene clusters will be evaluated for their antimicrobial activity as potential therapeutics.